Previous studies showed that most cases of ALK ؉ anaplastic large-cell lymphoma (ALK ؉ ALCL) do not express SHP1, a tyrosine phosphatase and an important negative regulator for cellular signaling pathways such as that of JAK/STAT. To fully assess the biologic significance of loss of SHP1 in ALK ؉ ALCL, we transfected SHP1 plasmids into 2 SHP1 ؊ , ALK ؉ ALCL cell lines, Karpas 299 and SU-DHL-1. After 24 hours of transfection, pJAK3 and pSTAT3 were decreased, and these changes correlated with downregulation of STAT3 downstream targets including cyclin D3, mcl-1, and bcl-2. Expression of SHP1 in these 2 cell lines also resulted in marked decreases in the protein levels of JAK3 and NPM-ALK, and these effects were reversible by proteosome inhibitor MG132. Conversely, when SHP1 expression in SUP-M2 (a SHP1 ؉ ALK ؉ ALCL cell line) was inhibited using siRNA, pSTAT3, pJAK3, JAK3, and NPM-ALK were all up-regulated. Coimmunoprecipitation studies showed that SHP1 was physically associated with JAK3 and NPM-

Summary JSI‐124 (cucurbitacin I) has been recently described as a specific inhibitor of signal transducer and activator of transcription‐3 (STAT3). As STAT3 activation is pathogenetically important in anaplastic lymphoma kinase‐positive anaplastic large cell lymphoma (ALK+ ALCL), we investigated whether JSI‐124 can mediate significant inhibitory effects in this cell type. In two ALK+ ALCL cell lines (Karpas 299 and SU‐DHL‐1), JSI‐124 significantly reduced the number of viable cells to 50% of that of negative controls at a dose of 5–10 μmol/l at 24 h and 1–1·25 μmol/l at 48 h. This decrease in viability was associated with apoptosis, as confirmed by the increase in the subG0/1 fraction, poly(ADP‐ribose)polymerase cleavage and expression of active caspase 3. JSI‐124 decreased the phosphorylated‐STAT3 and ‐Janus kinase‐3 (JAK3) levels in a dose‐dependent fashion, and these changes were coupled with significant decreases in several STAT3 downstream targets, including mcl‐1, bcl‐2, bcl‐xL and cyclin D3. Interestingly, JSI‐124 also dramatically decreased the protein levels of JAK3 and nucleophosmin (NPM)‐ALK, and these effects were reversible by MG132. Our data support that JSI‐124 is a potentially useful therapeutic agent for ALK+ ALCL. In addition to its role as a tyrosine kinase inhibitor, JSI‐124 appears to be involved in regulating proteosome degradation for proteins such as JAK3 and NPM‐ALK.

Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALK þ ALCL) is characterized by constitutive activation of the Janus kinase (JAK)3/signal transducers and activators of transcription 3 (STAT3) signaling pathway. SHP1, a tyrosine phosphatase that negatively regulates JAK/STAT, is frequently absent in ALK þ ALCL owing to gene methylation. To test the hypothesis that loss of SHP1 contributes to JAK3/ STAT3 activation in ALK þ ALCL cells, we induced SHP1 expression using 5-aza-2 0 -deoxycytidine (5-AZA), an inhibitor of DNA methyltransferase, in ALK þ ALCL cell lines, and correlated with changes in the JAK3/STAT3 pathway. 5-AZA gradually restored SHP1 expression in Karpas 299 and SU-DHL-1 cells over 5 days. The initially low level of SHP1 expression did not result in significant changes to the expression or tyrosine phosphorylation of JAK3 and STAT3. However, higher levels of SHP1 seen subsequently correlated with substantial decreases in JAK3 and pJAK3, followed by pSTAT3 (but not STAT3). Importantly, the decrease in JAK3 was abrogated by MG132, a proteasome inhibitor. 5-AZA induced no significant increase in apoptosis but it sensitized ALCL cells to doxorubicin-induced apoptosis. Our findings support the concept that loss of SHP1 contributes to the constitutive activation of JAK3/STAT3 in ALK þ ALCL cells. SHP1 appears to downregulate JAK3 by two mechanisms: tyrosine dephosphorylation and increased degradation via the proteasome pathway.

Mantle cell lymphoma is an aggressive B-cell lymphoma for which the biology is incompletely understood. Previous studies have reported that somatic hypermutation of the variable region of the immunoglobulin heavy chain gene (V H ), as commonly defined as o98% homology, can be detected in approximately one-third of mantle cell lymphoma, although the V H mutation status has not been found to significantly correlate with patient survival. In this study, we assessed V H mutation in 55 mantle cell lymphomas using a method slightly different from those used in the previous studies, and we came to different conclusions. Using DNA extracted from formalin-fixed/paraffin-embedded tumors in all cases, we identified monoclonal IGH bands in 54 of 55 cases with the FR1c/J H primer; a monoclonal IGH band was amplified using another IGH primer set, FR256/J H , in the remaining case. Cloning was performed in all cases, and an average of six clones were sequenced and analyzed for each case. Intraclonal heterogeneity was detected in 45 (82%) cases. Further analysis was performed in 53 cases, in which a predominant IGH species was identified. Most (32 of 53 cases, 60%) cases were 'mutated', with o98% homology. V H 1-69, V H 4-59 and V H 3-74 were utilized in 29 (55%) cases. Intraclonal evolution and nonproductive V H rearrangements were more frequent in the mutated group. Patients with the 'mutated' genotype had longer overall survival (P ¼ 0.017, Log rank) that is independent of the international prognostic index. To conclude, our data suggest that the V H mutation frequency in mantle cell lymphoma may be higher than previously believed. Importantly, using our methodology, we found that the V H mutation status may be a useful prognostic marker for these patients. Mantle cell lymphoma is a distinct clinicopathologic entity recognized by the World Health Organization Classification Scheme. 1 The genetic hallmark of this disease is the chromosomal abnormality, the t(11;14)(q13;q32), which results in cyclin D1 overexpression. 2 Using transgenic mouse models, two research groups have shown that enforced cyclin D1 expression in B cells is not sufficient for lymphomagenesis. 3,4 Recent studies, including a number of oligonucleotide array studies, revealed additional biochemical abnormalities in mantle cell lymphoma, such as defects in the apoptotic pathway, cell cycle progression and DNA repair. [5][6][7][8][9][10] To further understand the biology of this type of B-cell neoplasm, one approach is to examine the somatic hypermutation of the variable region of the immunoglobulin heavy chain (V H ) gene. In chronic lymphocytic leukemia, it has been shown that a subset of cases has relatively high levels of V H somatic mutation, and mutated cases were associated with better clinical outcome when using o98% homology as the cutoff. 11-14 More recently, several large studies were performed to examine the V H mutation status in mantle cell lymphoma; using o98% homology (compared to the germline sequences) as the cutoff, it was found that 29-34% of

No abstract

Background: Lymphomagenesis is believed to depend on clonal proliferation of neoplastic progenitor cells. For follicular lymphoma, the neoplastic progenitors are likely derived from the early B-cells, but their exact nature remains elusive. We report a case of transmission of a follicular lymphoma by allogeneic bone marrow transplantation. Follicular lymphoma developed in the donor, and subsequently in the recipient, years after the BMT. Testing supports a common progenitor cell for both the malignancies. Methods: Sequencing of the amplifiable translocation (14;18) BCL2/IgH fusion gene product and microsatellite analysis using a commercially available human identity testing kit were used to establish the biological relatedness of the donor and recipient lymphomas. The monoclonal IgH gene rearrangements from these two lymphomas were further examined for their somatic hypermutational status. Results: Microsatellite analysis confirmed the recipient’s lymphoma was donor cell-derived. Analysis of the monoclonal IgH gene rearrangements revealed post-germinal center cell phenotype in both tumors. The VH regions utilized were different in the donor and recipient lymphoma, demonstrating two distinct clonal populations. Despite this difference, the sequences of the translocation (14;18) fusion gene products (BCL2/IgH) from the two lymphomas were identical confirming a common progenitor cell for both malignancies. Conclusion: Our findings strongly suggest the same neoplastic progenitor cells are responsible for the lymphomagenesis in both the donor and recipient. These findings support the concept of a lymphoma progenitor cell.